Even under the most optimistic scenarios, global population is projected to continue growing well into the 21st century (UN, 2004), with the demand for meat production alone doubling between 1999 and 2050 (Steinfeld et al., 2006). One major obstacle to satisfying this demand is animal infection by internal helminth parasites, which are essentially ubiquitous in all farm animals globally.
Helminthic parasites are a polyphyletic group of worm-like animals including the taxa cestodes, nematodes, trematodes and monogeneans. Many helminthic parasites are spread via the digestive system, firstly via the ingestion of material contaminated with parasites or their eggs. During their life cycle such parasites may migrate to other parts of the body but return to the gut to produce more eggs which are then egested in the feces to provide a source for new infections.
Helminthic infection causes significant morbidity in animals, including humans, and, in the case of agricultural livestock, substantial economic loss due to reduced productivity from reduced weight gain, milk production and reproduction. Although severe infections can lead to death, ruminants carrying internal parasites, even at subclinical levels, exhibit a significant loss of productivity via a number of modalities—including anemia, decreased reproductive fitness and lactation, and reduction of food utilization—leading to reduced growth and poor body condition (Piedrafita et al., 2010; Perry and Randolph, 1999; Hoglund et al., 2001; Reinhardt et al., 2006). In the US, annual losses in livestock productivity have been estimated at three billion dollars annually (Bagley et al., 2014). In developing nations—where veterinary care, anthelmintic drugs, and regimented antihelminthic farming strategies are either less available or less widely practiced, and where food shortages are both more likely and more severe—the losses are expected to be substantially greater. Helminth infection is also an issue in agriculturally important non-ruminants, including poultry (Permin et al., 1999) and swine (Nansen and Roepstorff, 1999), and in other economically important herbivores such as horses (Duncan, 1985). In the case of horses, parasite infection also results in reduced physical performance of the animal. Internal parasite infestation, however, is not restricted to agricultural livestock and is also a problem for companion comfort animals such as cats and dogs. Infestation in cats and dogs can lead to a phenomenon called zoonosis resulting in hookworms and roundworms infecting humans' most often young children31.
Parasitic helminthic infections are also widespread in humans. Onchocerca volvulus, for example, causes onchocerciasis (also known as river blindness) in humans. About 17 to 25 million people throughout the world are reported to be infected with O. volvulus, with approximately a million people being having some amount of loss of vision. Brugia filariids can infect humans and other animals, causing diseases including filariasis (including lymphatic filariasis), elephantiasis and tropical eosinophilia. Schistosomiasis is a vascular parasitic disease in humans caused by blood flukes of the schistosoma species. Schistosomiasis is one of many helminthic diseases infecting over a billion people worldwide. These diseases include ascariasis, trichuriasis, enterobiasis, filariasis, trichinosis, onchocerciasis, fascioliasis, and cysticercosis. Schistosomiasis ranks second to malaria as a major cause of morbidity and suffering due to parasites.
Compounding the above problems, resistance to commonly used dewormers (anthelmintic resistance) is a global and growing problem in livestock production (Kaplan, 2004). Multidrug resistance is a common phenomenon in sheep and goat operations (da Cruz et al., 2010), with several cases of total anthelmintic failure reported (Cezar et al., 2010; Howell et al., 2008). Similarly, equine parasites have been documented to be resistant to all classes of dewormer currently available on the market, and the overwhelming majority of equine operations are facing resistance to at least one drug class (Peregrine et al., 2014). Most recently, cattle parasites have been found increasingly resistant to several classes of commonly used anthelmintic products (Gasbarre et al., 2009; Waghorn et al., 2006; Jackson et al., 2006).
Moreover, this resistance appears to be evolving at a significantly faster rate than that at which new dewormer products are being developed and launched: no new anthelmintic drug classes have been launched for use in large animals in North American since the early 1980s. For these reasons, veterinary organizations world-wide are promoting the monitoring of parasite infection and the presence of drug resistance as a critical aspect of animal care (Wood et al., 1995).
In the area of veterinary medicine, helminth infection is sometimes diagnosed by manifested clinical symptoms. The central tool in the practice of veterinary parasitology is the fecal egg-count (FEC), which has remained relatively unchanged for almost a century4, 11 and, in general, rely on the flotation of eggs in a sugar and/or salt medium that is denser than the eggs themselves, followed by microscopic examination and manual counting. Two FEC methods are currently in use.
The first, and perhaps the most universally used egg-counting procedure is the McMaster slide counting method, originally developed with sheep feces by Gordon and Whitlock in 1939. In this method, the fecal matter is suspended in a sugar and/or salt (SS) solution of greater density than the parasitic eggs themselves and placed in a microscopic slide especially fabricated for the purpose (the McMaster slide counting method). The eggs float to the surface (thereby separating them from the denser fecal debris to facilitate visualization) and are counted manually by a trained individual using a microscope at a 40-100× magnification.
A second, commonly used egg-counting procedure is the Wisconsin method and its derivatives. For these methods, eggs are floated directly, usually under the influence of a centrifugal field but also by gravity, onto a coverslip placed on the surface meniscus of the flotation medium. Sensitivity can be improved by sampling more of the fecal suspension by placing it in a tube to form a meniscus. The meniscus is overlaid with a coverslip and then subjected to centrifugation in a swing-out rotor (Rossanigo and Gruner, 1991; Egwang and Slocombe, 1981). Eggs adhering to the coverslip can then be counted microscopically as before.
While McMaster-type assays are simpler to perform, the high dilutions and small samplings involved result in assays with higher variability and lower sensitivity than Wisconsin-type tests. Conversely Wisconsin tests recover more of the eggs because of the increased flotation produced by centrifugal fields and also the large sample volumes they permit, but are technically slightly more demanding and require access to a centrifuge, which is impractical for many veterinary practices and in the field.
Unfortunately both of these techniques are not only time-consuming but also require the use of specialized laboratory equipment (i.e., a microscope, with or without a centrifuge), which is seldom available to veterinarians on-site, much less to the animal owners. Furthermore, the majority of animal owners do not possess the required training to reliably examine such samples (e.g., a layman might easily confuse the eggs with other fecal particulates such as pollen grains). Generally samples are collected and either sent to the veterinarian's office for analysis or to a third-party analytical laboratory, resulting in added cost and/or delays in diagnosis times. Alternatively, owners can ship the feces direct to laboratories via a number of commercial services but generally need to wait 48 hours for results. This, coupled to time-consuming nature of the tests and the requirement of trained personnel to process and inspect each sample, with a concomitant impact on cost, results in fewer animal owners routinely testing their livestock for the presence of parasites and the development of resistance.
In addition to the time and equipment issues, current egg-count methods suffer from a number of technical drawbacks. For example, egg-count variability limits the effectiveness of current egg-count methods. Equine egg counts have been estimated to vary by +/−50% between repeated counts (partially due to subjective inter-analyst variability, and partly for statistical reasons due to small sampling volumes). Thus, delineating between a true change and chance variability in fecal egg-count reduction is a real challenge (Vidyashankar et al., 2012). Although variability can be reduced by better analyst training, performing repeated counts, or using methods with lower detection limits, all of these solutions come with a cost in either additional training or processing time. Another drawback are egg loss rates. A certain percentage of eggs in a sample remains trapped in the fecal debris and does not make it to the flotation step. Depending on the technique and the operator, the egg loss rate has been found to vary between 30 and 60% (Vidyashankar et al., 2012). Importantly the detection limit, the lowest egg count detectable by the given technique, provides a technical limitation to the known methods. It is typically within the range of 1-50 eggs-per-gram (EPG). McMaster techniques (see FIG. 1) usually have detection limits of 25 or 50 EPG, making it very difficult to detect low egg count levels, which is particularly important for resistance testing (Vidyashankar et al., 2012).
As a result, prophylactic treatment with anthelmintic drugs has become standard across the industry. Unfortunately, and analogous to the over-prescription of antibiotics, the rising frequency of drug resistant nematodes is a growing global concern across all species, particularly small ruminants (Kaplan, 2004; Wolstenholme et al., 2004; Sutherland and Leathwick, 2011; Jackson and Coop, 2000; Roepstorff et al., 1987; Coles et al., 2003; Cernanska et al., 2006; Kornele et al., 2014). Both veterinary and regulatory organizations have now officially recognized this rapidly growing problem and have issued guidelines to (1) help monitor the growth of such resistance, and (2) attempt to curtail it by reducing the current indiscriminative use of both prescription and over-the-counter anthelmintic drugs (Wood et al., 1995; EMEA, 2006; FDA, 2014).
Unfortunately, monitoring for the development of resistance by observing Fecal Egg Count Reduction (FECR) involves the use of egg flotation methods, with the disadvantages described above8. Thus, these methods are unlikely to be widely used. To underscore this supposition, a recent survey of Kentucky thoroughbred farms showed that most respondents were aware of, and concerned about, the phenomenon of drug-resistant parasites; yet, over 70% were still deworming prophylactically (Robert et al., 2014). In addition, research from USDA has shown that 50-70% of cattle farmers agree that internal parasites have a significant economic impact on their operations (USDA, 2011b); yet, only 0.7% make use of any type of laboratory testing for this problem (USDA, 2010). In summary, the inconvenience of fecal egg counting appears to represent a significant barrier to the widespread adoption of a more targeted approach to the treatment and management of parasite infection.
While regulatory authorities in Europe have recognized the threat of the emergence drug resistant strains and moved to restrict their availability, in the United States many of these drugs are currently still openly available to the public over-the-counter. As a result, the use of fecal egg count reduction tests (FECRs) have been recommended by veterinary professional associations. FECRs depend upon systematically assessing parasite burden using egg counts as a surrogate marker of parasite load both before and after treatment with anti-helminthic drugs. Development of resistance can be detected by a lower-than-expected drop in egg counts after treatment, prompting appropriate managed responses to mitigate the proliferation of resistant strains through the population. As a result the fecal egg count has grown increasingly more important, while surprisingly very little progress has been made in improving this clinically important diagnostic tool.
Despite the passage of almost a century and the development of sophisticated modern analytical methodologies in other areas, the practice of egg counting has remained relatively unchanged, with the majority of innovations restricted to modifications in the flotation media or to methods for collecting eggs from larger fecal samples. For example, the volume of flotation chambers have been enlarged to improve sensitivity14,15,18, or alternative flotation solutions have been explored14,17.
Some workers have made efforts to develop more sophisticated efforts to either detect or quantify eggs in feces, using methods such as the Polymerase Chain Reaction (Demeler et al., 2013; Learmount et al., 2009) and flow cytometry (Colditz et al., 2002). However, such sophisticated techniques, along with the expertise and equipment required to conduct them, make them even more impractical as tools for anything other than research purposes.
Although some efforts have been made towards discovering egg-surface probes to aid in detection, these efforts have been limited to the screening of various lectins, in order to determine species of eggs to which these lectins can bind. Once determined, these proteins can be used as species-specific markers for the detection (but not quantification) of the presence of certain species or genera (Palmer and McCombe, 1996; Hillrichs et al., 2012; Colditz et al., 2002). However, because total egg count remains the standard for clinical decision making and for monitoring anthelmintic resistance, it remains to identify a marker that is generic for all helminth eggs. The identification of such a ubiquitous and experimentally tractable marker would open up the possibility of utilizing it to develop a rapid quantitative method to enumerate total fecal egg load and thus supplant flotation methods and their associated shortcomings.
Unfortunately, little work has been carried out to elucidate the molecular composition of clinically relevant egg surfaces, and what little that has been done has yielded little specific information (Wharton, 1983; Quiles et al., 2006).
Parasitic protozoal infections are also responsible for a wide variety of diseases of medical and veterinary importance, ranging across malaria and Pneumocystis carinii pneumonia in man and various coccidioses in birds, fish and mammals. Many of the diseases are life threatening to the host and cause considerable economic loss in animal husbandry. Coccidiosis is a protozoan parasitic disease that affects the intestinal tract of animals including, but not limited to, cattle, sheep, goats, rabbits, pigs, chickens, turkeys, cats, and dogs. Parasites of interest include, but are not limited to Isospora sp. (dogs and cats); Eimeria maxima, E. acervulina, E. brunetti, E. necatrix, and E. tenella (chickens); E. meleagridis, E. gallopavonis, E. adenoeides, and E. dispersa (turkeys); E. bovis (cattle); E. ovina (sheep); E. porci (pigs); and E. stiedai (rabbits). Coccidiosis is one of the most economically important diseases in many livestock species. The disease is characterized by diarrhea, unthriftiness, loss of appetite and weight, and variable levels of mortality. In lifestock animals, economic losses are caused by a decreased weight gain due in part to the malabsorption of nutrients through the gut wall. And, as with helminth infections, the state of the art for detecting protozoan oocysts relies on time-consuming methods.
While many parasites spend part of their lifecycle in the gastrointestinal tract and are egested in feces, many other parasites infect the bladder and kidneys the host animal. Pearsonema plica and Dioctophyme renale are two such parasites known to infect the bladder and kidney in dogs. Eggs of both genera can be found in urin samples. Schistosomiasis species may infect the urinary tract or intestines, the eggs of which may be found in the animal's urine or stool.
As can be seen, although more modern methods have been developed, these tests have not been widely adopted and manual egg counting has remained the most widely available and routinely used method to monitor internal parasite infestation (i.e., helminth and protozoan parasite infestation). Thus, a need exists for a simple, accurate and cost-effective method for quantifying the number of parasite eggs in feces that can be performed by either veterinarians in the field or by animal owners themselves.